加压CO_2杀菌机理初探及对乳中酶和细菌活性的影响
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摘要
加压CO2杀菌是指利用高于或接近于临界温度(31.1℃)和临界压力(7.13MPa)的CO2对食品进行杀菌的技术。CO2是一种天然、安全的杀菌剂,加压CO2杀菌技术的杀菌温度低,杀菌后可通过减压使CO2完全分离出来,被认为是一种非常有前途的冷杀菌技术。本课题的目的是首先研究加压CO2对生理盐水中细菌的杀菌效果、并分析影响因素和残存细菌活性；然后研究加压CO2的杀菌机理；再对比研究加压CO2与巴氏杀菌对原料乳天然菌群的杀菌效果及在贮藏期间天然菌群的生长情况；最后研究CO2对原料乳中蛋白酶和脂肪酶活性的影响及在贮藏期间酶活性的改变情况。将加压C02杀菌技术应用到乳品工业将是一项技术革新,通过本课题研究将为加压CO2冷杀菌技术提供理论依据,对使用加压CO2对乳及乳制品杀菌具有重要意义。
使用加压CO2处理生理盐水中的大肠杆菌、荧光假单胞菌、枯草芽孢杆菌和金黄色葡萄球菌。通过改变压力(1.5MPa、3MPa、4.5MPa、6MPa和7.5MPa)、时间(20min、40min、60min、80min和100min)、温度(30℃、35℃、40℃、45℃和50℃)、加压介质(生理盐水、脱脂乳和全脂乳)和加压方式(连续加压40min、间歇加压20min×2和间歇加压10min×4)研究影响杀菌效果的因素同时研究残存细菌活性的改变情况(30℃、7.5MPa、加压CO2连续处理20min、40min、60min)。结果表明加压CO2的杀菌效果随着压力、时间和温度提高而逐渐提高；牛乳成分对细菌具有保护作用；在低压下改变加压方式并不能显著(P>0.05)提高杀菌效果；加压CO2对4种菌的杀菌效果为：荧光假单胞菌>大肠杆菌>枯草芽孢杆菌>金黄色葡萄球菌；对革兰氏阴性菌的杀菌效果要好于革兰氏阳性菌；加压CO2对残存大肠杆菌和荧光假单胞菌具有后续破坏作用,使残存细菌的生长迟滞期发生显著延长(P<0.05),但对残存枯草芽孢杆菌和金黄色葡萄球菌并没有产生明显后续破坏作用,残存细菌的生长迟滞期并没有发生延长。
使用加压CO2处理生理盐水中的大肠杆菌(7.5MPa,处理温度为25℃和45℃,处理时间为10min、20min、30min、40min、50min)和枯草芽孢杆菌(7.5MPa,处理温度为25℃和45℃,处理时间为20min、40min、60min、80min、100min)。与巴氏杀菌(63℃、30min)比较,通过检测细菌细胞膜通透性的改变情况,菌体内蛋白质、核酸、K+和Mg2+的泄漏情况,菌体超微结构和菌体DNA的改变情况,用FTIR分析菌体成分和菌体蛋白质二级结构的改变情况来综合研究加压CO2的杀菌机理。结果表明当加压CO2处理使菌体致死的过程中,细胞膜通透性显著(P<0.05)变大,但与热杀菌不同,没有发生全透改变；短时加压CO2处理不足以使菌体内蛋白质泄漏,长时加压CO2处理致使蛋白泄漏,并且泄漏量是显著(P<0.05)大于巴氏杀菌,但所需时间比99%以上菌体死亡时间明显滞后,所以只是细菌死亡后的后发结果；加压CO2处理使菌体内核酸发生显著(P<0.05)泄漏,菌体死亡与核酸泄漏有关；菌体死亡与加压CO2处理引起的菌体K+和Mg2+泄漏有关；加压CO2处理使菌体发生变形,破坏了菌体细胞壁和细胞膜,由此也证明菌体细胞膜通透性变大,菌体内成分发生泄漏。加压CO2处理后,菌体DNA没有发生明显降解,说明菌体死亡与菌体DNA损伤无关；通过FTIR分析可知,加压C02处理导致菌体脂肪酸发生改变,核酸物质发生改变,大肠杆菌的肽聚糖层发生明显改变,枯草芽孢杆菌的肽聚糖层变化并不明显,大肠杆菌的蛋白质性质发生改变,枯草芽孢杆菌的蛋白质性质发生不规律改变；与加热处理不同,C02处理使大肠杆菌蛋白质的α-螺旋结构减少,转变为β-折叠结构和β-转角结构,枯草芽孢杆菌蛋白质的β-折叠结构增加,α-螺旋结构、β-转角结构、无规卷曲结构的改变没有规律。
使用加压C02处理原料乳。研究加压C02与巴氏杀菌对原料乳中天然菌群的杀灭效果以及对比研究了在贮藏期间处理后原料乳的稳定性。通过改变C02压力(4.5、7.5、10.5MPa)、加压时间(20、40、60min)、温度(4、15、25℃)和加压方式(连续加压和间歇加压)研究加压C02处理参数对杀菌效果的影响。在最适条件下在冷藏温度下加压C02处理对于原料乳中天然菌群的贮藏稳定性的影响也被研究,并且被比较和巴氏杀菌样品(63℃,30min)。结果表明原料乳中假单胞菌、肠杆菌科细菌、乳酸菌和金黄色葡萄球菌菌数降低,细菌总数也降低；C02压力、时间、温度对杀菌效果影响较大,加压方式的影响相对较小；对于加压C02处理原料乳中革兰氏阳性菌比革兰氏阴性菌更加具有抵抗力；在7.5MPa、4℃下,连续加压C02处理40min,原料乳中的金黄色葡萄球菌菌数降低到不能检测的水平,假单胞菌、肠杆菌科细菌和细菌总数降低到低于巴氏杀菌的水平,乳酸菌菌数高于巴氏杀菌水平；在贮藏期间,CO2处理原料乳中的假单胞菌、肠杆菌科细菌菌数和细菌总数低于巴氏杀菌,乳酸菌菌数高于巴氏杀菌,未检测出金黄色葡萄球菌：与巴氏杀菌相比,C02处理使原料乳的保藏期延长2天。
使用加压C02处理原料乳(压力7.5MPa,温度25℃和45℃,时间20min和40min,连续加压)。结果表明与未处理原料乳蛋白酶活性3.61U/mL和脂肪酶活性0.43U/mL相比,CO2处理后不脱气时,CO2处理原料乳的蛋白酶活性(不同条件下分别为2.92U/mL、2.92U/mL2.40U/mL、2.20U/mL)和脂肪酶活性(不同条件下分别为0.25U/mL、0.26U/mL、0.16U/mL、0.12U/mL)都发生显著(P<0.05)降低；CO2处理后脱气时,原料乳的蛋白酶活性(不同条件下分别为3.30U/mL、3.42U/mL、3.24U/mL、3.21U/mL)发生较小(P>0.05)回升,脂肪酶活性(不同条件下分别为0.55U/mL.0.51U/mL、0.57U/mL、0.52U/mL)发生显著(P<0.05)提高；加压CO2处理能够抑制原料乳在贮藏过程中的蛋白质分解,著且短时加压的抑制作用更大,但温度对抑制作用的影响并不大；加压CO2处理导致原料乳在贮藏过程中脂肪分解程度变大,并且低温和长时加压的脂肪分解程度更大,脂肪分解加速是在加压CO2处理条件下原料乳的脂肪球变小所致。
Pressurized CO2treatment was a food sterilization technology that using the temperature above or close the critical temperature of31.1℃and the pressure above or close the critical pressure7.13MPa. CO2was a natural and safe antiseptic. The sterilization temperature of pressurized CO2technology was low. CO2could be separated completely by decompression after sterilization. Pressurized CO2treatment was considered as a very promising cold sterilization. The purpose of this project is to investigate the bactericidal effects of pressurized CO2on the bacteria in saline, analyse the influencing factors and the residual bacterial activity; investigate the sterilization mechanism of pressurized CO2; compare the sterilization effects of pressurized CO2and thermal pasteurization on natural microorganisms in raw milk and the growth of natural microorganisms during storage; investigate the effects of pressurized CO2on protease and lipase activity in raw milk and the change of activity during storage. The application of pressurized CO2sterilization techniques to the dairy industry would be a technological innovation, the research of this subject would provide the theoretical basis for CO2cold sterilization technology, and the research of this subject had the significant meaning to the pressurized CO2sterilization of milk and dairy products.
Escherichia coli, Pseudomonas fluorescens, Bacillus subtilis and Staphylococcus aurens in saline were treated by pressurized CO2. The factors influencing the bactericidal effects were studied by changing the pressure (1.5MPa,3MPa,4.5MPa,6MPa and7.5MPa), time (20min,40min,60min,80min and100min), temperature (30℃,35℃,40℃,45℃and50℃), media (saline, skim milk and whole milk) and modus (40min,20min×2and10min×4). The change of residual bacterial activity was also studied (30℃,7.5MPa, treated by pressurized CO220min,40min and60min). The results showed that bactericidal effects of pressurized CO2were increased when the pressure, time, temperature were increased. The milk ingredients could protect the bacteria. Changing the pressurized modus under low pressure could not increase the bactericidal effects. The bactericidal effects of pressurized CO2to the four bacteria were Pseudomonas fluorescens> Escherichia coli> Bacillus subtilis> Staphylococcus aureus. The bactericidal effects of pressurized CO2to Gram-negative bacteria were better than Gram-positive bacteria. Pressurized CO2had the subsequent damaging effects to residual Escherichia coli and Pseudomonas fluorescens, the growth lag phases of residual bacteria were extended a lot. But pressurized CO2did not have the subsequent damaging effects to residual Bacillus subtilis and Staphylococcus aureus, the growth lag phases of residual bacteria were not extended.
Escherichia coli (7.5MPa,25℃/45℃,10min/20min/30min/40min/50min) and Bacillus subtilis (7.5MPa,25℃/45℃,20min/40min/60min/80min/100min) in saline were treated by pressurized CO2Compared with pasteurization (63℃,30min), the sterilization mechanism of pressurized CO2was studied by detecting the change of membrane permeability, the leakage of bacterial protein, nucleic acid, K+and Mg2+, the change of bacterial ultrastructure, the change of bacterial DNA. The change of bacterial components and bacterial protein secondary structure were also studied by FTIR. The results showed that the membrane permeability was increased (P<0.05) when the bacteria were inactivated by pressurized CO2, but differently from thermal pasteurization, the transmissive changes were not happened. Protein leakage was not happened under pressurized CO2treatment of short time. Although the protein leakage under pressurized CO2treatment of long time was more than (P<0.05) thermal pasteurization significantly, the time was lagged to the time of99%bacterial death. So protein leakage under pressurized CO2treatment of long time was not the reason of bacterial death, was only the secondary phenomenon of bacterial death. Pressurized CO2treatment induced the bacterial nucleic acid leaked significantly (P<0.05), so bacterial death was related to the bacterial nucleic acid leakage. Bacterial death was related to the bacterial K+and Mg2+leakage induced by pressurized CO2treatment. Pressurized CO2treatment destroyed the cell wall and membrane, bacterial deformation was happened. It was also certified that the bacterial membrane permeability was increased and the bacterial components were leaked. The degradation of bacterial DNA was not happened after pressurized CO2treatment, so bacterial death was not related to the damage of bacterial DNA. It was known through FTIR that pressurized CO2treatment induced the change of bacterial fatty acid and nucleic acid. The peptidoglycan layer of Escherichia coli changed obviously, the peptidoglycan layer of Bacillus subtilis did not change obviously. The protein of Escherichia coli changed obviously, the protein of Bacillus subtilis changed erratically. Differently from thermal pasteurization, pressurized CO2treatment induced the a-helix structure of Escherichia coli decreased, the a-helix structure changed to the β-sheet structure and the β-turn structure. The β-sheet structure of Bacillus subtilis increased, the change of a-helix structure, p-turn structure and the random coil structure of Bacillus subtilis were irregular.
Raw milk was treated by pressurized CO2. The inactivation effects of pressurized CO? and pasteurization on natural microorganisms in raw milk and the stability of the treated raw milk during storage were investigated comparatively. The effects of pressurized CO2treatment parameters on the inactivation results were investigated by changing CO2pressure (4.5,7.5and10.5MPa), pressurized time (20,40and60min), temperature (4,15and25℃) and pressurized modus (continuous treatment and intermittent treatments). The effect of pressurized CO2treatment at the optimal conditions on the storage stability of natural microorganisms in raw milk at4℃was investigated and compared with the samples pasteurized at63℃for30min. The results showed that the viable counts of Pseudomonas, Enterobacteriaceae, LAB and Staphylococcus aureus in raw milk were decreased and the total bacterial count was also decreased. The CO2pressure, pressurized time and temperature had more profound effects on the inactivation degree while the effect of pressurized modus was less profound relatively. The gram-positive bacteria in raw milk were more resistant to the pressurized CO2treatment than gram-negative bacteria. At7.5MPa,4℃, under continuous pressurized CO2treatment for40min, the viable count of Staphylococcus aureus was decreased to the level undetectable, the viable counts of Pseudomonas, Enterobacteriaceae and the total bacterial count were decreased to the levels lower than pasteurization, and the viable count of LAB was more than pasteurization. During storage, the viable counts of Pseudomonas, Enterobacteriaceae and the total bacterial count in the samples treated by pressurized CO2were lower than which in the samples treated by pasteurization, the viable count of LAB was more, Staphylococcus aureus was undetectable. Compared with pasteurization, the storage time of raw milk was extended two days.
Raw milk was treated by pressurized CO2(7.5MPa,25℃/45℃,20min/40min, continuous treatment). The results showed that compared with the protease activity of untreated raw milk3.61U/mL and the lipase activity of untreated raw milk0.43U/mL, when not degassed after pressurized CO2treatment, the protease activity (2.92U/mL,2.92U/mL,2.40U/mL,2.20U/mL) and lipase activity (0.25U/mL,0.26U/mL,0.16U/mL,0.12U/mL) of pressurized CO2treated raw milk were decreased significantly (P<0.05), when degassed after pressurized CO2treatment, the protease activity (3.30U/mL,3.42U/mL,3.24U/mL,3.21U/mL) of raw milk was increased slightly (P>0.05) and the lipase activity (0.55U/mL,0.51U/mL,0.57U/mL,0.52U/mL) was increased significantly (P<0.05). Pressurized CO2treatment could inhibit the proteolytic of raw milk during storage, and the inhibition effect was more when under short time, but the effect of temperature was not more. Pressurized CO2treatment induced the lipolysis of raw milk increased during storage, and the degree of lipolysis was more under long time and low temperature. The smaller fat globules induced by pressurized CO2treatment was the reason of the speeded-up of lipolysis.
使用加压CO2处理生理盐水中的大肠杆菌、荧光假单胞菌、枯草芽孢杆菌和金黄色葡萄球菌。通过改变压力(1.5MPa、3MPa、4.5MPa、6MPa和7.5MPa)、时间(20min、40min、60min、80min和100min)、温度(30℃、35℃、40℃、45℃和50℃)、加压介质(生理盐水、脱脂乳和全脂乳)和加压方式(连续加压40min、间歇加压20min×2和间歇加压10min×4)研究影响杀菌效果的因素同时研究残存细菌活性的改变情况(30℃、7.5MPa、加压CO2连续处理20min、40min、60min)。结果表明加压CO2的杀菌效果随着压力、时间和温度提高而逐渐提高；牛乳成分对细菌具有保护作用；在低压下改变加压方式并不能显著(P>0.05)提高杀菌效果；加压CO2对4种菌的杀菌效果为：荧光假单胞菌>大肠杆菌>枯草芽孢杆菌>金黄色葡萄球菌；对革兰氏阴性菌的杀菌效果要好于革兰氏阳性菌；加压CO2对残存大肠杆菌和荧光假单胞菌具有后续破坏作用,使残存细菌的生长迟滞期发生显著延长(P<0.05),但对残存枯草芽孢杆菌和金黄色葡萄球菌并没有产生明显后续破坏作用,残存细菌的生长迟滞期并没有发生延长。
使用加压CO2处理生理盐水中的大肠杆菌(7.5MPa,处理温度为25℃和45℃,处理时间为10min、20min、30min、40min、50min)和枯草芽孢杆菌(7.5MPa,处理温度为25℃和45℃,处理时间为20min、40min、60min、80min、100min)。与巴氏杀菌(63℃、30min)比较,通过检测细菌细胞膜通透性的改变情况,菌体内蛋白质、核酸、K+和Mg2+的泄漏情况,菌体超微结构和菌体DNA的改变情况,用FTIR分析菌体成分和菌体蛋白质二级结构的改变情况来综合研究加压CO2的杀菌机理。结果表明当加压CO2处理使菌体致死的过程中,细胞膜通透性显著(P<0.05)变大,但与热杀菌不同,没有发生全透改变；短时加压CO2处理不足以使菌体内蛋白质泄漏,长时加压CO2处理致使蛋白泄漏,并且泄漏量是显著(P<0.05)大于巴氏杀菌,但所需时间比99%以上菌体死亡时间明显滞后,所以只是细菌死亡后的后发结果；加压CO2处理使菌体内核酸发生显著(P<0.05)泄漏,菌体死亡与核酸泄漏有关；菌体死亡与加压CO2处理引起的菌体K+和Mg2+泄漏有关；加压CO2处理使菌体发生变形,破坏了菌体细胞壁和细胞膜,由此也证明菌体细胞膜通透性变大,菌体内成分发生泄漏。加压CO2处理后,菌体DNA没有发生明显降解,说明菌体死亡与菌体DNA损伤无关；通过FTIR分析可知,加压C02处理导致菌体脂肪酸发生改变,核酸物质发生改变,大肠杆菌的肽聚糖层发生明显改变,枯草芽孢杆菌的肽聚糖层变化并不明显,大肠杆菌的蛋白质性质发生改变,枯草芽孢杆菌的蛋白质性质发生不规律改变；与加热处理不同,C02处理使大肠杆菌蛋白质的α-螺旋结构减少,转变为β-折叠结构和β-转角结构,枯草芽孢杆菌蛋白质的β-折叠结构增加,α-螺旋结构、β-转角结构、无规卷曲结构的改变没有规律。
使用加压C02处理原料乳。研究加压C02与巴氏杀菌对原料乳中天然菌群的杀灭效果以及对比研究了在贮藏期间处理后原料乳的稳定性。通过改变C02压力(4.5、7.5、10.5MPa)、加压时间(20、40、60min)、温度(4、15、25℃)和加压方式(连续加压和间歇加压)研究加压C02处理参数对杀菌效果的影响。在最适条件下在冷藏温度下加压C02处理对于原料乳中天然菌群的贮藏稳定性的影响也被研究,并且被比较和巴氏杀菌样品(63℃,30min)。结果表明原料乳中假单胞菌、肠杆菌科细菌、乳酸菌和金黄色葡萄球菌菌数降低,细菌总数也降低；C02压力、时间、温度对杀菌效果影响较大,加压方式的影响相对较小；对于加压C02处理原料乳中革兰氏阳性菌比革兰氏阴性菌更加具有抵抗力；在7.5MPa、4℃下,连续加压C02处理40min,原料乳中的金黄色葡萄球菌菌数降低到不能检测的水平,假单胞菌、肠杆菌科细菌和细菌总数降低到低于巴氏杀菌的水平,乳酸菌菌数高于巴氏杀菌水平；在贮藏期间,CO2处理原料乳中的假单胞菌、肠杆菌科细菌菌数和细菌总数低于巴氏杀菌,乳酸菌菌数高于巴氏杀菌,未检测出金黄色葡萄球菌：与巴氏杀菌相比,C02处理使原料乳的保藏期延长2天。
使用加压C02处理原料乳(压力7.5MPa,温度25℃和45℃,时间20min和40min,连续加压)。结果表明与未处理原料乳蛋白酶活性3.61U/mL和脂肪酶活性0.43U/mL相比,CO2处理后不脱气时,CO2处理原料乳的蛋白酶活性(不同条件下分别为2.92U/mL、2.92U/mL2.40U/mL、2.20U/mL)和脂肪酶活性(不同条件下分别为0.25U/mL、0.26U/mL、0.16U/mL、0.12U/mL)都发生显著(P<0.05)降低；CO2处理后脱气时,原料乳的蛋白酶活性(不同条件下分别为3.30U/mL、3.42U/mL、3.24U/mL、3.21U/mL)发生较小(P>0.05)回升,脂肪酶活性(不同条件下分别为0.55U/mL.0.51U/mL、0.57U/mL、0.52U/mL)发生显著(P<0.05)提高；加压CO2处理能够抑制原料乳在贮藏过程中的蛋白质分解,著且短时加压的抑制作用更大,但温度对抑制作用的影响并不大；加压CO2处理导致原料乳在贮藏过程中脂肪分解程度变大,并且低温和长时加压的脂肪分解程度更大,脂肪分解加速是在加压CO2处理条件下原料乳的脂肪球变小所致。
Pressurized CO2treatment was a food sterilization technology that using the temperature above or close the critical temperature of31.1℃and the pressure above or close the critical pressure7.13MPa. CO2was a natural and safe antiseptic. The sterilization temperature of pressurized CO2technology was low. CO2could be separated completely by decompression after sterilization. Pressurized CO2treatment was considered as a very promising cold sterilization. The purpose of this project is to investigate the bactericidal effects of pressurized CO2on the bacteria in saline, analyse the influencing factors and the residual bacterial activity; investigate the sterilization mechanism of pressurized CO2; compare the sterilization effects of pressurized CO2and thermal pasteurization on natural microorganisms in raw milk and the growth of natural microorganisms during storage; investigate the effects of pressurized CO2on protease and lipase activity in raw milk and the change of activity during storage. The application of pressurized CO2sterilization techniques to the dairy industry would be a technological innovation, the research of this subject would provide the theoretical basis for CO2cold sterilization technology, and the research of this subject had the significant meaning to the pressurized CO2sterilization of milk and dairy products.
Escherichia coli, Pseudomonas fluorescens, Bacillus subtilis and Staphylococcus aurens in saline were treated by pressurized CO2. The factors influencing the bactericidal effects were studied by changing the pressure (1.5MPa,3MPa,4.5MPa,6MPa and7.5MPa), time (20min,40min,60min,80min and100min), temperature (30℃,35℃,40℃,45℃and50℃), media (saline, skim milk and whole milk) and modus (40min,20min×2and10min×4). The change of residual bacterial activity was also studied (30℃,7.5MPa, treated by pressurized CO220min,40min and60min). The results showed that bactericidal effects of pressurized CO2were increased when the pressure, time, temperature were increased. The milk ingredients could protect the bacteria. Changing the pressurized modus under low pressure could not increase the bactericidal effects. The bactericidal effects of pressurized CO2to the four bacteria were Pseudomonas fluorescens> Escherichia coli> Bacillus subtilis> Staphylococcus aureus. The bactericidal effects of pressurized CO2to Gram-negative bacteria were better than Gram-positive bacteria. Pressurized CO2had the subsequent damaging effects to residual Escherichia coli and Pseudomonas fluorescens, the growth lag phases of residual bacteria were extended a lot. But pressurized CO2did not have the subsequent damaging effects to residual Bacillus subtilis and Staphylococcus aureus, the growth lag phases of residual bacteria were not extended.
Escherichia coli (7.5MPa,25℃/45℃,10min/20min/30min/40min/50min) and Bacillus subtilis (7.5MPa,25℃/45℃,20min/40min/60min/80min/100min) in saline were treated by pressurized CO2Compared with pasteurization (63℃,30min), the sterilization mechanism of pressurized CO2was studied by detecting the change of membrane permeability, the leakage of bacterial protein, nucleic acid, K+and Mg2+, the change of bacterial ultrastructure, the change of bacterial DNA. The change of bacterial components and bacterial protein secondary structure were also studied by FTIR. The results showed that the membrane permeability was increased (P<0.05) when the bacteria were inactivated by pressurized CO2, but differently from thermal pasteurization, the transmissive changes were not happened. Protein leakage was not happened under pressurized CO2treatment of short time. Although the protein leakage under pressurized CO2treatment of long time was more than (P<0.05) thermal pasteurization significantly, the time was lagged to the time of99%bacterial death. So protein leakage under pressurized CO2treatment of long time was not the reason of bacterial death, was only the secondary phenomenon of bacterial death. Pressurized CO2treatment induced the bacterial nucleic acid leaked significantly (P<0.05), so bacterial death was related to the bacterial nucleic acid leakage. Bacterial death was related to the bacterial K+and Mg2+leakage induced by pressurized CO2treatment. Pressurized CO2treatment destroyed the cell wall and membrane, bacterial deformation was happened. It was also certified that the bacterial membrane permeability was increased and the bacterial components were leaked. The degradation of bacterial DNA was not happened after pressurized CO2treatment, so bacterial death was not related to the damage of bacterial DNA. It was known through FTIR that pressurized CO2treatment induced the change of bacterial fatty acid and nucleic acid. The peptidoglycan layer of Escherichia coli changed obviously, the peptidoglycan layer of Bacillus subtilis did not change obviously. The protein of Escherichia coli changed obviously, the protein of Bacillus subtilis changed erratically. Differently from thermal pasteurization, pressurized CO2treatment induced the a-helix structure of Escherichia coli decreased, the a-helix structure changed to the β-sheet structure and the β-turn structure. The β-sheet structure of Bacillus subtilis increased, the change of a-helix structure, p-turn structure and the random coil structure of Bacillus subtilis were irregular.
Raw milk was treated by pressurized CO2. The inactivation effects of pressurized CO? and pasteurization on natural microorganisms in raw milk and the stability of the treated raw milk during storage were investigated comparatively. The effects of pressurized CO2treatment parameters on the inactivation results were investigated by changing CO2pressure (4.5,7.5and10.5MPa), pressurized time (20,40and60min), temperature (4,15and25℃) and pressurized modus (continuous treatment and intermittent treatments). The effect of pressurized CO2treatment at the optimal conditions on the storage stability of natural microorganisms in raw milk at4℃was investigated and compared with the samples pasteurized at63℃for30min. The results showed that the viable counts of Pseudomonas, Enterobacteriaceae, LAB and Staphylococcus aureus in raw milk were decreased and the total bacterial count was also decreased. The CO2pressure, pressurized time and temperature had more profound effects on the inactivation degree while the effect of pressurized modus was less profound relatively. The gram-positive bacteria in raw milk were more resistant to the pressurized CO2treatment than gram-negative bacteria. At7.5MPa,4℃, under continuous pressurized CO2treatment for40min, the viable count of Staphylococcus aureus was decreased to the level undetectable, the viable counts of Pseudomonas, Enterobacteriaceae and the total bacterial count were decreased to the levels lower than pasteurization, and the viable count of LAB was more than pasteurization. During storage, the viable counts of Pseudomonas, Enterobacteriaceae and the total bacterial count in the samples treated by pressurized CO2were lower than which in the samples treated by pasteurization, the viable count of LAB was more, Staphylococcus aureus was undetectable. Compared with pasteurization, the storage time of raw milk was extended two days.
Raw milk was treated by pressurized CO2(7.5MPa,25℃/45℃,20min/40min, continuous treatment). The results showed that compared with the protease activity of untreated raw milk3.61U/mL and the lipase activity of untreated raw milk0.43U/mL, when not degassed after pressurized CO2treatment, the protease activity (2.92U/mL,2.92U/mL,2.40U/mL,2.20U/mL) and lipase activity (0.25U/mL,0.26U/mL,0.16U/mL,0.12U/mL) of pressurized CO2treated raw milk were decreased significantly (P<0.05), when degassed after pressurized CO2treatment, the protease activity (3.30U/mL,3.42U/mL,3.24U/mL,3.21U/mL) of raw milk was increased slightly (P>0.05) and the lipase activity (0.55U/mL,0.51U/mL,0.57U/mL,0.52U/mL) was increased significantly (P<0.05). Pressurized CO2treatment could inhibit the proteolytic of raw milk during storage, and the inhibition effect was more when under short time, but the effect of temperature was not more. Pressurized CO2treatment induced the lipolysis of raw milk increased during storage, and the degree of lipolysis was more under long time and low temperature. The smaller fat globules induced by pressurized CO2treatment was the reason of the speeded-up of lipolysis.
引文
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